Diakou et al. Parasites Vectors (2019) 12:519 https://doi.org/10.1186/s13071-019-3760-9 Parasites & Vectors

SHORT REPORT Open Access Efcacy of a moxidectin/imidacloprid spot‑on formulation ­(Advocate®) for the treatment of Troglostrongylus brevior in naturally infected cats in a feld study in Greece Anastasia Diakou1, Simone Morelli2, Dimitris Dimzas1, Angela Di Cesare2, Gioia Capelli3, Chiara Parrinello2, Matthias Pollmeier4, Roland Schaper4 and Donato Traversa2*

Abstract Background: Troglostrongylus brevior is a lungworm of wild felids that recently has been recognized as agent of severe respiratory disease in domestic cats in Mediterranean and Balkan countries. Nevertheless, the information on treatment options for feline troglostrongylosis is still poor. The aim of this pilot feld trial was to evaluate the efcacy of the spot-on formulation containing 1% w/v moxidectin and 10% w/v imidacloprid (Advocate­ ® spot-on solution for cats, Bayer Animal Health GmbH) in the treatment of T. brevior infection in naturally infected cats in Greece. Methods: The trial was a negative control, multicentre, clinical efcacy study conducted according to the standards of Good Scientifc Practice (GSP). Sixteen cats in two study sites, naturally infected with T. brevior, were allocated to an untreated control group (G1, n 8) or a treatment group (G2, n 8), according to a randomization list. Animals = = assigned to G2 were treated with ­Advocate® for cats on days 0 and 28 at the recommended dose rate and animals assigned to G1 received a rescue treatment with the same product on days 56 and 84. Efcacy was assessed on days 28 and 56 in G2 and on days 84 and 112 in G1 by faecal larval counts. The primary efcacy criterion was the absence of T. brevior frst-stage larvae (L1) following treatment. Other efcacy parameters were the quantitative comparison of L1 presence before (baseline) and after one or two treatments in both groups. Results: All G2 cats were negative for T. brevior L1 at the frst post-treatment evaluation (100% efcacy) while G1 cats were persistently shedding L1. The diference of the mean number of L1 per gram between G2 and G1 was statisti- cally signifcant (P < 0.001). All G1 cats were negative (100% efcacy) for T. brevior L1 at the frst post-rescue-treatment evaluation. Therefore, treatment efcacy at study completion was 100% in both groups in terms of stopping the L1 shedding in the faeces of the animals. No adverse efects were observed during the study. Conclusions: These results indicate that ­Advocate® spot-on solution for cats represents an option for treating cats naturally infected with T. brevior. Keywords: Troglostrongylus brevior, Cat, Moxidectin, Treatment

*Correspondence: [email protected] 2 Faculty of Veterinary Medicine, University of Teramo, Località Piano D’Accio snc., 64100 Teramo, Italy Full list of author information is available at the end of the article

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Background Methods Troglostrongylus brevior, a parasitic nematode that Study design afects bronchi and bronchioles of wild felids (e.g. Felis Te study (BAH Study No.: 205121) was conducted silvestris silvestris), has recently attracted scientifc inter- according to the standards of good scientifc practice est, due to its ability to induce severe bronchopneu- (GSP) and the National Animal Welfare requirements, monia in domestic cats and its apparent spreading in with the approval of the National Organisation of Med- various countries [1]. Te parasite has an indirect life- icines of Greece (No. 25953). Te study was a nega- cycle involving a gastropod (terrestrial slugs or snails) as tive control, multicentre, partially blinded (examining intermediate host, in which the frst-stage larvae, shed investigator blinded) trial, following a randomized by the infected cat, develop into the infective third larval block design. stage (L3). Paratenic hosts (birds, rodents, reptiles) prob- ably play a key role in the biological cycle, facilitating the transmission of the parasite to the defnitive host [2]. A Pre‑inclusion screening vertical route of transmission is also known although Between study days (SD)-18 and SD-10, faecal samples it is unclear if it occurs via the utero or/and the milk of cats under null or irregular antiparasitic treatments [3]. Troglostrongylus brevior infection in cats has been for endoparasites, referred to private veterinary prac- increasingly reported since 2010 in diferent countries tices located in seven study areas, i.e. two already known of Europe, i.e. Spain, Italy, Greece, Cyprus and Bulgaria, as enzootic (Mykonos and Attica) [7] and fve with no with a prevalence ranging from 1.2% to 14% [4]. In the previous relevant information (Rhodes, Xanthi, Kavala, vast majority of cases, this lungworm has been described Tessaloniki and Syros), were subjected to qualitative in areas where the natural host of T. brevior, the Euro- Baermann’s examination. Parasite morphological features pean wildcat, is present, although infected domestic cats were identifed using published keys [1, 12]. have been found also in regions where the presence of From the cats that were found positive for T. brevior, a wildcats is not documented [4, 5]. faecal sample was collected between SD-6 and SD-3 and Cat troglostrongylosis has been proven severe and life- subjected to a quantitative Baermann test as previously threatening in kittens and young animals, as it can cause described [13, 14] to confrm the infection and determine irreversible lung damage and is a potential threat for the the baseline numbers of L1 larvae per gram of faeces litter in the case of pregnancy or lactation [1–4, 6, 7]. (LPG). Te identity of the L1 microscopically identifed To date, the only product licensed for the treatment as T. brevior was confrmed with a species-specifc PCR of T. brevior infection in cats is a spot-on formulation [6]. containing eprinomectin in combination with fpronil, (S)-methoprene and praziquantel ­(Broadline™ spot- on solution for cats, Boehringer Ingelheim, Ingelheim Inclusion and exclusion criteria and allocation to study am Rhein, Germany). Other anthelmintics containing groups emodepside or moxidectin have recently shown their On SD 0 the potentially enrollable cats were subjected potential efectiveness in treating T. brevior infection to veterinary examination and body weighing. Only ani- in pilot studies [8], in clinical settings [9] or in experi- mals that satisfed the following criteria were included ® mental studies on intermediate hosts [10]. ­Advocate in the study: (i) cats shedding at least 15 LPG of T. bre- spot-on solution for cats (Bayer Animal Health GmbH, vior between SD-7 and SD 0; (ii) cats ≥ 9 weeks of age Leverkusen, Germany) is a combination of 1% w/v mox- and weighing at least 1 kg; (iii) cats with no severe clin- idectin and 10% w/v imidacloprid, licensed globally for ical signs at physical examination on SD 0; (iv) cats for treatment and prevention of a variety of ecto- and endo- which none of the restrictions of the product label was parasites. In Europe, this product is labelled for feline applicable; (v) cats without lesions in the application area cardio-pulmonary and intestinal nematodes for preven- (base of the neck) of the investigational veterinary prod- tion (Diroflaria immitis) and/or treatment (Capillaria uct (IVP); (vi) cats for which the owner provided written aerophila, Toxocara cati and Ancylostoma tubaeforme) owner consent form. [11]. More recently it has also been labelled for preven- Animals for which the following criteria were con- tion and treatment of Aelurostrongylus abstrusus. Given frmed were excluded from the study: (i) cats showing preliminary data that have indicated the potential util- severe clinical signs compatible with T. brevior infec- ity of moxidectin in treating T. brevior infection in cats tion, for which it was estimated that the inclusion in the ® [9, 10], this study investigated the efcacy of ­Advocate study might have permanently compromised health; (ii) against T. brevior in naturally infected cats in enzootic pregnant or lactating cats, or cats for which mating was areas of Greece. planned within the next 16 weeks; (iii) cats with known Diakou et al. Parasites Vectors (2019) 12:519 Page 3 of 5

hypersensitivity to at least one of the ingredients of the examination. A secondary efcacy endpoint has been set I V P. based on the reduction of the LPG values from the pre- All cats meeting the inclusion and not meeting the treatment assessment (baseline) to SD 28/29 (post-treat- exclusion criteria were included in the study and ran- ment collection 1) and 56/57 (post-treatment collection domly allocated to two study groups, i.e. Group 1 (G1) 2) in cats of G2, calculated as percent reduction accord- left untreated (n = 11 cats) and Group 2 (G2) (n = 8 cats) ing to the formula: treated with ­Advocate®. (Pre - treatment mean LPG −Post - treatment mean LPG) Reduction (% ) = × 100 Pre - treatment mean LPG

Anthelmintic treatment A further efcacy criterion was the LPG values reduc- Cats assigned to the G2 were treated twice at a monthly tion from baseline to SDs 83/84 and 111/112 in cats of interval on SD 0 and SD 28/29. To generate additional G1, calculated as above. efcacy information and for ethical reasons G1 cats Te signifcance of the diference of the mean LPG received a rescue treatment twice at a monthly interval between the two groups at each time point was assessed with ­Advocate® on SD 56/57 and SD 83/84. All treat- through analysis of variance (ANOVA), using the soft- ments were administered by the collaborating vet- ware SPSS for Windows, version 13.0. erinarians at the recommended dose rate of ≥ 10 mg imidacloprid/kg body weight (BW) and 1.0 mg mox- ≥ Results idectin/kg BW corresponding to ≥ 0.1 ml spot-on formu- lation per kg BW. Cats and pre‑treatment evaluation All cats were closely monitored by the veterinarian for Of the overall 336 cats examined for T. brevior L1 shed- 1 hour after each administration of the product to record ding, 19 (5.6%), i.e. 18 from Mykonos and 1 from Rho- and eventually treat any occurring adverse efect. Te des, were selected for the study based on the inclusion owners were advised to refer the animal to the veterinary criteria. clinic in the case of any unusual sign. Tree of the cats initially allocated in G1, were with- drawn from the study as 2 of them were lost and 1 died Post‑treatment evaluation in a car accident. Te fnal study population (n = 16, 8 in Cats of both groups were examined on SDs 28/29 and G1 and 8 in G2) was composed of 9 (56.25%) female and 7 56/57 assessing the post-treatment LPG value with by (43.75%) male cats with age ranging from 7 to 36 months quantitative Baermann examination. Cats of G1 were and a weight ranging from 1.8 to 5 kg. All the animals additionally evaluated on SDs 83/84 and 111/112. completed the study according to the protocol, i.e. on SD Te primary efcacy criterion was the absence of 56/57 for G2 and on SD 111/112 for G1, except for one cat T. brevior L1 on SD 28/29 and SD 56/57 (post-treat- in G1 that was not presented for the fnal evaluation on ment) in G2 cats, evaluated at the quantitative Baermann SD 111/112 and therefore completed the study on SD 84. At baseline, study cats had an average of 31.88 (stand- ard deviation, sd = 36.247, 95% confdence interval, CI:

Table 1 Pre- and post-treatment larval faecal shedding of cats included in the study to evaluate the efcacy of Advocate® in the treatment of natural troglostrongylosis Study day Group n Mean LPG SD SE 95% CI Min-Max ANOVA

0 G1 8 31.88 36.247 12.815 1.57–62.18 15–120 F 0.468; P = 0.505 G2 8 46.88 50.351 17.802 4.78–88.97 15–165 = 28 G1 8 20.63 7.763 2.745 14.13–27.12 15–30 F 56.467; = G2 8 0 0 0 0 0 P < 0.001* 56 G1 8 18.75 6.944 2.455 12.94–24.56 15–30 F 58.333; = G2 8 0 0 0 0 0 P < 0.001*

Abbreviations: G1, cats treated with Advocate on study days 56/57 and 83/84; G2, cats treated with Advocate on study days 0 and 28/29; n, number of animals included in G1 or G2; LPG, larvae per gram; CI, confdence interval; SD, standard deviation; SE, standard error; Min, minimum; Max, maximum *Statistically signifcant results Diakou et al. Parasites Vectors (2019) 12:519 Page 4 of 5

T. brevior after one (87.5%) or two (100%) administrations 1.57–62.18) and 46.88 (sd = 50.351, CI: 4.78–88.97) LPG in G1 and G2, respectively (Table 1). two weeks apart in terms of reduction of larval shedding. Additionally, in the latter study the majority of sympto- Efcacy evaluation matic cats recovered clinically after the frst treatment, All of the cats enrolled in G2 were negative at the quan- except for one of the cats for which a second administra- titative Baermann examination performed at SD 28/29 tion was necessary for a complete clinical recovery [8]. and SD 56/57. Te cats enrolled in G1 were persistently Until now, spot on moxidectin has been administered infected with an average of 20.63 and of 18.75 LPG at SD in a few cats infected with T. brevior. For instance, a single ® 28/29 and SD 56/57 respectively (Table 1). Diferences in administration of ­Advocate was 100% efective in reduc- mean LPG values were statistically signifcant (P < 0.001) at ing the larval shedding in two kittens with monospecifc all post-treatment examinations (Table 1). All cats enrolled infection by T. brevior, with one of these kittens clinically in G1 resulted negative at the quantitative Baer­mann recovering after the single spot-on application, while the examination performed on SD 83/84 and SD 111/112. other showed clinical and radiographic signs up to four Te animal that was not presented for examination on SD weeks post-treatment before reaching a complete recov- 112 was however negative at SD 83. Terefore, treatment ery, probably due to an initial more severe clinical condi- with ­Advocate® showed an efcacy of 100% in eliminating tion [9]. In the same study, two repeated administrations, T. brevior L1 shedding after a single treatment. No adverse two weeks apart, were necessary for the elimination of lar- efects were recorded after each administration of the vae shed by three cats infected with both A. abstrusus and product and throughout the study period. T. brevior [9]. In a study focusing on larval infectivity in mollusc intermediate hosts, spot-on moxidectin also Discussion proved highly efcacious in stopping T. brevior larval Te present results show that spot-on moxidectin con- shedding in cats after a single administration [10]. How- tained in Advocate® is efcacious and safe in treating ever, in a fatal case of troglostrongylosis in a kitten, the T. brevior infections under natural conditions. administration of moxidectin spot-on did not succeed Troglostrongylus brevior is the agent of a severe and [18], most likely due to the general condition of the ani- emerging parasitic disease with an often fatal outcome in mal at admission and the severe lung lesions caused by kittens [5, 6]. However, subclinical infections also occur the parasite. Tus, a prompt diagnosis and a timely and [6] as in the case of cats enrolled in the present study. Te efective treatment are crucial, especially because pre- diagnosis of T. brevior infection in cats can be challeng- ventive options against T. brevior infection are currently ® ing due to overlapping clinical features with various feline unavailable. Nonetheless, a certain efcacy of Advocate­ respiratory diseases, i.e. bacterial, viral or fungal infec- in preventing T. brevior has been recently discussed in a tions or other parasitic diseases (i.e. aelurostrongylosis study that has shown that cats living in areas enzootic for or capillariosis) [1, 6, 9]. A prompt etiological diagnosis, T. brevior and subjected to known risk factors (i.e. preying, that would allow an immediate and efective therapeutic outdoor habitat) were negative for this lungworm when ® approach, is therefore of primary importance. Further- under a chemopreventive scheme with ­Advocate applied more, if not treated quickly, troglostrongylosis can lead for D. immitis [19]. Moreover, with regard to kittens, given ® to permanent damages, such as irreversible pulmonary that Advocate cannot be administered to cats under nine hypertension and related chronic complications, thus weeks of age, the application of preventive treatments of compromising the quality of life of infected cats [4, 15]. pregnant cats could potentially protect the kittens from Given the emergence of T. brevior in cats and the sub- the vertical transmission. Nevertheless, it should be taken ® sequent increased awareness towards this parasite, the in account that ­Advocate could be administered to preg- interest on the efcacy of parasiticides has increased. nant cats only according to a beneft-risk assessment and Apart from the licensed spot-on formulation contain- under the supervision of a responsible veterinarian [11]. ing eprinomectin (Broadline™, spot-on solution for cats, Boehringer Ingelheim) [16, 17] oral milbemycin oxime Conclusions has also been proved potentially efcacious in treating Te present data demonstrate the 100% efcacy of ® cats infected with T. brevior either in monospecifc or Advocate­ in terms of stopping larval shedding in cats natu- in mixed infection with A. abstrusus, in terms of larval rally infected with T. brevior. Te cessation of L1 excretion shedding and complete clinical recovery [9]. However, was achieved already with a single administration 28/29 these data were obtained from only a few cats and further days post-treatment in all study animals. Furthermore, no investigations are needed [9]. Another trial [8] proved adverse efects were recorded in any of the cats included in ® that the spot-on formulation containing emodepside in the trial. ­Advocate acts on a variety of endo- and ectopara- combination with praziquantel is highly efective against sites, with a broad spectrum that is advantageous especially Diakou et al. Parasites Vectors (2019) 12:519 Page 5 of 5

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Single and genaki, Sapfo Chatzoudi, Manolis Vorrisis, Ismini Drakouli, Melpomeni Tagalidi, mixed feline lungworm infections: clinical, radiographic and therapeutic Evaggelia Kontostoli and Alexandros Makaronas for their invaluable contribu- features of 26 cases (2013–2015). J Feline Med Surg. 2017;19:1017–29. tion in the present study. 10. Cavalera MA, Colella V, Napoli E, Arfuso F, Panarese R, Brianti E, Otranto D. Shedding of feline lungworm larvae and their infectivity to snail intermedi- Authors’ contributions ate hosts after anthelmintic treatment. Int J Parasitol. 2019;49:449–53. MP, ADC and DT prepared the study design and coordinated the whole study. 11. EMA (European Medicines Agency). Product information Advocate - SM, DD and ADC participated in the clinical, treatment and laboratory activi- EMEA/V/C/000076 - II/0039/G. 2018. https​://www.ema.europ​a.eu/en/medic​ ties of the study. AD and DT coordinated and supervised the local project ines/veter​inary​/EPAR/advoc​ate. Accessed 15 May 2019. activities in Greece. CP participated in the laboratory activities. GC performed 12. Brianti E, Giannetto S, Dantas-Torres F, Otranto D. Lungworms of the genus the statistical analysis. MP and RS participated in monitoring the study and Troglostrongylus (Strongylida: Crenosomatidae): neglected parasites for supervising paper drafting. All authors assisted in the manuscript preparation. domestic cats. Vet Parasitol. 2014;202:104–12. All authors read and approved the fnal manuscript. 13. Traversa D, Di Cesare A, Milillo P, Lohr B, Iorio R, Pampurini F, et al. Efcacy and safety of imidacloprid 10%/moxidectin 1% spot-on formulation in Funding the treatment of feline aelurostrongylosis. Parasitol Res. 2009;105(Suppl. This study was funded by Bayer Animal Health GmbH. 1):S55–62. 14. Traversa D, Milillo P, Di Cesare A, Lohr B, Iorio R, Pampurini F, et al. Efcacy Availability of data and materials and safety of emodepside 2.1% praziquantel 8.6% spot-on formulation in All data generated or analyzed during this study are included in this published the treatment of feline aelurostrongylosis. Parasitol Res. 2009;105(Suppl. article. 1):S83–9. 15. Crisi PE, Traversa D, Di Cesare A, Luciani A, Civitella C, Santori D, Boari A. Ethics approval and consent to participate Irreversible pulmonary hypertension associated with Troglostrongylus brevior The present study was authorized by National Organisation of Medicines of infection in a kitten. Res Vet Sci. 2015;102:223–7. Greece (No. 25953). All study cats were privately owned naturally infected cats 16. Giannelli A, Brianti E, Varcasia A, Colella V, Tamponi C, Di Paola G, et al. for which each owner signed a consent form and accepted to participate in Efcacy of Broadline® spot-on against Aelurostrongylus abstrusus and the study. According to the Greek and European legislation, and as confrmed Troglostrongylus brevior lungworms in naturally infected cats from Italy. Vet by the Greek Authorities for Animal Welfare (decision 311340/2166), no license Parasitol. 2015;209:273–7. related to the welfare of animals was required for the conduction of the trial. 17. Giannelli A, Capelli G, Joachim A, Hinney B, Losson B, Kirkova Z, et al. Lungworms and gastrointestinal parasites of domestic cats: a European Consent for publication perspective. Int J Parasitol. 2017;47:517–28. Not applicable. 18. Brianti E, Gaglio G, Giannetto S, Annoscia G, Latrofa MS, Dantas-Torres F, et al. Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Creno- Competing interests somatidae) as agents of broncho-pulmonary infestation in domestic cats. MP and RS are currently employed by Bayer Animal Health GmbH. AD, SM, DD, Parasit Vectors. 2012;5:178. ADC, GC, CP and DT declare that they have no competing interests. 19. Morelli S, di Frangipane Regalbono A, Russi I, Crisi PE, Simonato G, Pampurini F, Traversa D. Efcacia preventiva di un prodotto a base di imidacloprid e Author details moxidectina nei confronti dei parassiti cardio-respiratori di cane e gatto. 1 Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Summa Veterinaria. 2018;5:33–8. Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. 2 Faculty of Veterinary Medicine, University of Ter- amo, Località Piano D’Accio snc., 64100 Teramo, Italy. 3 Istituto Zooproflattico Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- Sperimentale delle Venezie, Legnaro, Padova, Italy. 4 Bayer Animal Health lished maps and institutional afliations. GmbH, Leverkusen, Germany.

Received: 10 June 2019 Accepted: 22 October 2019